PROJECT SUMMARY: The overarching goal of this proposal is to uncover a previously uncharacterized molecular function of c-Myc (hereafter referred to as Myc), a proto-oncoprotein that is frequently amplified in breast cancer and many other types of human cancer. Recently, we found that Myc overexpression leads to elevated expression of glutamate-ammonia ligase (GLUL) and interestingly, this Myc-induced GLUL is not through the direct transactivation by Myc, rather it involves promoter demethylation of the GLUL gene. We further found that the demethylation is dependent on increased expression of thymine DNA glycosylase (TDG), which is a direct Myc transcriptional target. These results suggest an unexpected role of Myc in promoting glutamine synthesis, and intriguingly, suggest a previously unidentified molecular function of Myc in activating gene expression by regulating DNA methylation. This prompts us to form the hypothesis that Myc can regulate gene expression via the modulation of DNA methylation. We propose two Specific Aims to study this hypothesis. In Aim 1, we plan to identify Myc-induced DNA methylation and gene expression profiles by whole genome bisulfite sequencing (WGBS) and RNA-Seq using various breast cancer cell lines with stable or inducible expression or knock-down of Myc. We will first prepare cell cultures with different treatments and extract genomic DNA and total cellular RNA for the next-generation sequencing (NGS). After NGS sequence reads are obtained, we will perform the bioinformatics analysis to (1) identify and annotate differentially methylated regions (DMR) in WGBS, with focus on gene promoters; (2) identify differentially expressed genes (DEG) in RNA-seq, using the same samples for DMR; and (3) rank Myc ?epigenetic targets? using integrated bioinformatics analysis of DMR and DEG, and identify potential biological pathways preferentially affected by Myc through epigenetic regulation. We expect to discover specific ?epigenetic targets? of Myc in various breast cancer cell lines. In Aim 2, we plan to validate the identified methylation profiles using traditional molecular techniques and examine their biological relevance. We will first use the quantitative PCR and focal bisulfate sequencing on specific gene promoters to validate the ?epigenetic targets? targets of Myc to be found in Aim 1. We will also examine the expression of TDG and the Myc epigenetic targets in various breast cancer cell lines. Furthermore, we will examine the expression patterns of TDG and validated epigenetic Myc targets by IHC using de-identified breast cancer clinical tissue samples, and correlate them with histopathological characteristics and clinical outcomes. If successful, this project will uncover DNA demethylation as a novel mechanism for Myc regulated gene expression and oncogenesis. In the long run, the knowledge gained from this study will help with the understanding of cancer etiology and shed light on the development of novel therapeutics.